A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis. A centrifuge is generally driven by an electric motor. The centrifuge works using the sedimentation principle, where the centripetal acceleration causes more dense substance to separate out along the radial direction (the bottom of the tube). Simultaneously, lighter objects will tend to move to the top of the tube.
Protocols for centrifugation typically specify the amount of acceleration to be applied to the sample, rather than specifying a rotational speed such as revolutions per minute. This distinction is important because two rotors with different diameters running at the same rotational speed will subject samples to different accelerations. During circular motion the acceleration is the product of the radius and the square of the angular velocity and it is traditionally named “relative centrifugal force” (RCF). The acceleration is measured in multiples of “g” (or ×“g”), the standard acceleration due to gravity at the earth's surface, and it is given byRCF=r(2πN)2/g where g represents the earth's gravitational acceleration,
r represents the rotational radius, and
N represents the rotational speed, measured in revolutions per unit of time. The relationship may be written asRCF=1.118×10−5rcmN2RPM where rcm represents the rotational radius measured in centimeters,
NRPM represents the rotational speed measured in revolutions per minute (RPM)
g=(980.65 cm/sec2)(3600 sec2/minute2)=3,530,340 cm/minute2.
Clinical chemistry is the area of pathology that is generally concerned with analysis of body fluids. The discipline originated in the late nineteenth century with the use of simple chemical tests for various components of blood and urine. Subsequently other techniques were applied including the use and measurement of enzyme activities, spectrophotometry, electrophoresis, and immunoassay. Most current laboratories are not highly automated and use assays that are monitored closely and controlled for quality. Tests that require examination and measurement of the cells of blood, as well as blood clotting studies, are not included, as they are generally grouped under hematology. Clinical chemistry tests can be performed on any kind of body fluid, but are generally performed on serum or plasma. Serum is the yellow watery part of blood that is left after blood has been allowed to clot and all blood cells have been removed. Such removal is most easily done by centrifugation, which packs the denser blood cells and platelets to the bottom of the centrifuge tube, leaving the liquid serum fraction resting above the packed cells. Plasma is essentially the same as serum, but is obtained by centrifuging the blood without clotting. Plasma therefore contains all of the clotting factors, including fibrinogen.
An immunoassay is a biochemical test that measures the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed using immunoassay methods. Such assays are based on the unique ability of an antibody to bind with high specificity to one or a very limited group of molecules. A molecule that binds to an antibody is called an antigen. Immunoassays can be carried out for either member of an antigen/antibody pair. For antigen analytes, an antibody that specifically binds to that antigen can frequently be prepared for use as an analytical reagent. When the analyte is a specific antibody, its cognate antigen can be used as the analytical reagent. In either case the specificity of the assay depends on the degree to which the analytical reagent is able to bind to its specific binding partner to the exclusion of all other substances that might be present in the sample to be analyzed. In addition to the need for specificity, a binding partner must be selected that has a sufficiently high affinity for the analyte to permit an accurate measurement. The affinity requirements depend on the particular assay format that is used.
In addition to binding specificity, the other key feature of all immunoassays is a means for producing a measurable signal in response to a specific binding. Historically, the signal involved measuring a change in some physical characteristic such as light scattering or changes in refractive index. Most immunoassays today depend on the use of an analytical reagent that is associated with a detectable label. A large variety of labels have been demonstrated including radioactive elements used in radioimmunoassay; enzymes; fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; dye crystallites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals and others. Such labels serve for detection and quantitation of binding events either after separating free and bound labeled reagents or by designing the system in such a way that a binding event effects a change in the signal produced by the label. Immunoassays requiring a separation step, often called separation immunoassays or heterogeneous immunoassays, are popular because they are easy to design, but they frequently require multiple steps including careful washing of a surface onto which the labeled reagent has bound. Immunoassays in which the signal is affected by binding can often be run without a separation step. Such assays can frequently be carried out simply by mixing the reagents and sample and making a physical measurement. Such assays are called homogeneous immunoassays or less frequently non-separation immunoassays.
Regardless of the method used, interpretation of the signal produced in the immunoassay requires reference to a calibrator that mimics the characteristics of the sample medium. For qualitative assays the calibrators may consist of a negative sample with no analyte and a positive sample having the lowest concentration of the analyte that is considered detectable. Quantitative assays require additional calibrators with known analyte concentrations. Comparison of the assay response of a real sample to the assay responses produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample.
Hematology is the branch of internal medicine, physiology, pathology, clinical laboratory work, and pediatrics that is concerned with the study of blood, the blood-forming organs, and blood diseases. Hematology includes the study of etiology, diagnosis, treatment, prognosis, and prevention of blood diseases. The laboratory work that does into the study of blood is frequently performed by a medical technologist. Blood diseases affect the production of blood and its components, such as blood cells, hemoglobin, blood proteins, the mechanism of coagulation, etc.
Hematology tests include a wide variety of laboratory studies, ranging from coagulation factors to various cell evaluations. A sample of whole blood is taken, usually from a vein. Amounts differ according to the number and types of tests to be run and the testing instruments to be used. Typically red blood cells are counted and lysed (broken down); then white blood cells are measured. Because blood clots quickly, the measured blood sample is diluted with either a lysing agent or an anti-clotting agent, depending on the test(s) to be completed. A lysing agent breaks down the red blood cells and allows counting of white blood cells. Dilution is an important step in preparing samples for testing for several reasons. First, concentrations of the anticoagulant must be adequate for the volume of blood. Insufficient dilution may allow formation of small clots that lower cell counts; excessive dilution can cause cells to shrink or swell. Anticoagulants that are widely used are EDTA (ethylenediaminetetraacetic acid) and heparin. EDTA is used often for routine cell counts and platelet counts. Heparin inhibits clotting without distorting red blood cell volume and is the preferred anticoagulant for studies of leukocytes. Second even relatively large blood samples do not provide a sufficient quantity to flow through an analyzer for measurement. Blood must be mixed with a diluent that will allow the cells to be evenly suspended in sufficient liquid to flow at a constant rate for measurement. Counts and other tests should be run within three to four hours of obtaining blood samples (within one to two hours for platelet counts).
Automated hematology has been used in large laboratories for many years. Automated technologies include the following:
1. Impedance principle: This is the coulter principle based on the fact that blood cells are poor conductors of electricity. Coulter counters count cells as they flow, in single file, through an aperture in an electric field. As each cell crosses the aperture, the increase in electrical impedance is proportional to the cell's size. The cells are uniformly suspended in a diluent, and the suspension passes through the aperture at a constant rate. Because these counters discriminate between particles according to size, their thresholds (usually aperture size) can be set to the preferred size limit, depending on the cells to be counted. Dilutions for white blood cell count are critical. Because these cells are small and numerous, samples with high white blood cell counts may exceed the counting capacity of the instrument or may cause high coincidence counts—that is counts in which two cells pass through the aperture simultaneously. An increased amount of diluent may be needed to avoid these errors, or an instrument may automatically correct for these errors.
2. Optical principles: Automated systems are based on flow-through (also called flow cytometer) optical technologies that identify cells on the basis of their fluorescent labeling ability. Blood cells in a diluent pass through an aperture, scattering a focused light beam. Cells diffract (scatter) light in a manner that is measurable and unique to each cell type. Accuracy can be influenced by coincidence errors—two cells passing through the aperture may be measured as one large cell (primary coincidence) or two particles that are below the measurement threshold may be counted as one cell (secondary coincidence). In most cases, the coincidence factor is too small to distort the count significantly. Most instruments automatically correct for coincidence errors.
Automated analyzers for clinical chemistry that are commercially available include those sold under the trademarks “ARCHITECT” c16000, “ARCHITECT” c4000, and “ARCHITECT” c8000, all of which are commercially available from Abbott Laboratories, Abbott Park, Ill. Automated analyzers for immunoassays that are commercially available include those sold under the trademarks “ARCHITECT” i1000SR, “ARCHITECT” i2000SR, “ARCHITECT” i4000SR, and “AxSYM”, all of which are commercially available from Abbott Laboratories, Abbott Park, Ill. Automated analyzers for hematology that are commercially available include those sold under the trademarks “CELL-DYN” 1800, “CELL-DYN” 3200, and “CELL-DYN” 3700, all of which are commercially available from Abbott Laboratories, Abbott Park, Ill.
Automated analyzers for clinical chemistry, automated analyzers for immunoassays, and automated analyzers for hematology typically require one or more of the following components:
(a) Piercing station, where stoppers of sample tubes are penetrated to enable a sample aspirating probe to aspirate a portion of the sample;
(b) Automated system for transporting samples for moving samples from one automated analyzer to another, thereby integrating automated analyzers;
(c) Aspirating/dispensing devices for removing samples from sample tubes and dispensing samples into reaction vessels;
(d) Blood separation device for separating blood cells from plasma after a sample of whole blood has been withdrawn from a sample container;
(e) Decapping devices for removing caps from sample containers.
U.S. Pat. No. 6,033,355 describes a centrifuge having a plurality of individual centrifuge devices and a robotic loading and/or unloading device. Each of the centrifuge devices may be loaded and unloaded without affecting the operation of the other centrifuge devices. The centrifuge devices include a plurality of rotors that re spaced apart and translated along a predetermined path. The rotors are rotatably mounted with respect to the predetermined path and the rotational planes of the rotors extend substantially at right angles to the predetermined path.
Several other types of whole blood separation techniques are available, such as, for example, batch type centrifugation and serum filters.
Testing blood samples in a laboratory exposes operators to biological hazards, including aerosols. In addition, when operators remove stoppers from closed sample tubes, repetitive motion can lead to physical injuries, such as carpal tunnel syndrome. It would be desirable to develop a system to reduce these risks to the operators without reducing throughput and assay performance. It would also be desirable to reduce exposure of operators to cutting “sharps” hazards.
Laboratories that test blood samples expend substantial labor to sort sample tubes for hematology or immunoassay/clinical chemistry testing. In addition to this sorting, balancing and centrifugation is performed on sample tubes destined for immunoassay/clinical chemistry testing. These operations add a minimum of 10 minutes or more to time needed to obtain a result, and could delay an assay of a STAT sample by an additional 10 minutes. Integration of hematology/immunoassay/clinical chemistry testing would reduce the labor required for sorting, balancing, and centrifugation and significantly reduce the time needed to obtain a result by integrating the blood separation step into the analyzer system/work cell. It would be desirable to provide a fully automated method for processing one sample tube for hematology/immunoassay/clinical chemistry testing, while providing high throughput for large laboratories that process many samples, and random and continuous access assay processing, as well as analysis of STAT samples. In addition, it would be desirable to realize these benefits with the current product lines without requiring new analyzers to be developed. It would be desirable to develop systems for use in a variety of customer environments, e.g., laboratories, with minimal changes.
Patients and phlebotomists are required to provide two sample tubes for hematology testing and immunoassay/clinical chemistry testing. The integration of hematology/immunoassay/clinical chemistry testing would reduce patient distress, reduce cost of collecting samples, reduce cost of inventory (to stock more than one type of sample tube), and reduce cost of disposal of solid waste.
Designs for centrifuge have typically been “batch” type designs, requiring all sample tubes to be loaded and then separated for 10 minutes (at minimum). If a STAT sample arrives just after the centrifugation process begins, the STAT sample would be delayed 10 minutes (at minimum). A continuous access centrifuge would eliminate “batches”, and any subsequent delays for processing STAT samples, thereby reducing the time required for centrifugation to five (5) minutes.
Designs for centrifuge have typically been targeted for laboratories in developed countries, where electrical power, air conditioning, etc., are commonplace. A continuous access centrifuge would provide the ability receive energy from sources other than electrical, such as hand cranks, bicycles, windmills, waterwheels, etc., thereby allowing modern centrifugation techniques to be performed in developing countries.
Testing blood samples in a laboratory requires a substantial amount of labor to sort sample tubes for hematology testing, immunoassay testing, or clinical chemistry testing. In addition to this sorting, balancing and centrifugation is carried out for sample tubes destined for immunoassay testing and clinical chemistry testing. Balancing and centrifugation adds a minimum of 10 minutes or more to time needed to obtain a result.